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UNIVERSITY  OF  ILLINOIS 

Agricultural  Experiment  Station. 


BULLETIN    NO.   94. 


NITROGEN   BACTERIA  AND  LEGUMES 

(WiTH  SPECIAL  REFERENCE  TO  RED  CLOVER,  COWPEAS,  SOY  BEANS, 
ALFALFA,  AND  SWEET  CLOVER,  ON  ILLINOIS  SOILS). 


BY  CYRIL  G.  HOPKINS. 


Work  and  Knowledge  are  a  stronger  team  than  Work  and  Work. 


URBANA,  ILLINOIS,  FEBRUARY,  1904. 


SUMMARY  OF  BULLETIN  No.  94. 

1.  Soil  nitrogen  cannot  be  used  by  plants  until  it  is  changed  to  the  form  of 
nitrate  nitrogen  by  the  nitrifying  bacteria.  Page  307 

2.  Atmospheric  nitrogen  cannot  be  used  by  any  agricultural  plants,  excepting 
legumes,  and  even  leguminous  plants  have  no  power  to  obtain  nitrogen  from  the 
air  unless  they  are  provided  with  the  proper  nitrogen-gathering  bacteria.     Page  309 

3.  As  a  rule  each  important  agricultural  legume  must  have  its  own  particular 
species  of  bacteria.  Page  311 

4.  The  frequent  failure  of  red  clover  in  normal  seasons,  especially  on  normal 
soils  occupying  the  highest  land,  is  undoubtedly  due  in  part  at  least  to  the  absence 
of  the  proper  bacteria  (sometimes  the  soil  lacks  lime  or  phosphorus).       Page  313 

5.  On  the  very  acid  soils,  where  clover  has  never  been  grown  successfully, 
applications  of  ground  limestone  should  be  made  where  legumes  are  to  be  grown. 

Page  315 

6.  Cowpeas  need  not  be  inoculated,  because  the  cowpea  bacteria  are  usually 
either  present  in  the  soil  or  are  introduced  with  the  seed  in  sufficient  numbers  to 
effect  a  good  degree  of  infection  if  the  soil  is  suitable  and  if  cowpeas  are  seeded 
upon  the  same  land  for  two  successive  years.  Page  317 

7.  Cowpeas  grown  on  infected  soil  on  the  University  of  Illinois  soil  experiment 
field  contained  four  times  as  much  nitrogen  as  the  same  kind  of  cowpeas  grown 
on  similar  land  which  was  not  infected.  Page  319 

8.  As  a  rule  soy  bean  fields  should  be  inoculated  when  first  seeded  to  soy  beans, 
otherwise  they  may  be  grown  on  the  same  land  for  three  or  four  years  before  the 
soil  becomes  thoroughly  infected.  Page  320 

9.  Investigations,  reported  in  this  bulletin,  furnish  conclusive  proof  that  in- 
fected sweet  clover  soil  can  be  used  for  inoculating  alfalfa  fields  and  with  the  same 
results  as  ara  obtained  when  the  infected  soil  is  obtained  from  an  old  alfalfa  field. 
(Sweet  clover  is  a  tall,  rank-growing,  sweet-scented  leguminous  plant  widely  dis- 
tributed over  Illinois,  especially  along  the  roadsides  in  the  northern  and  central  parts 
of  the  state,  and  in  many  places  in  bottom  lands  in  southern  Illinois.)        Page  324 

10.  This  bulletin  will  be  sent  free  of  charge  to  any  one  interested  in  Illinois 
agriculture  upon  request  to  E.  Davenport,  Director  Agricultural  Experiment  Sta- 
tion, Urbana,  Illinois ;  and,  if  so  requested,  the  name  of  the  applicant  will  be  placed 
upon  the  permanent  mailing  list  of  the  Experiment  Station,  so  that  all  bulletins 
will  be  sent  to  him  as  they  are  issued. 

Conclusions  Page  327 


306 


NITROGEN    BACTERIA  AND    LEGUMES 

(WITH  SPECIAL  REFERENCE  TO  RED  CLOVER,  COWPEAS,  SOY  BEANS, 
ALFALFA,  AND  SWEET  CLOVER,  ON  ILLINOIS  SOILS). 

BY  CYRIL  G.  HOPKINS,  CHIEF  IN  AGRONOMY  AND  CHEMISTRY. 

Among  the  several  different  classes  or  groups  of  bacteria  there  are 
two  which  are  of  special  importance  to  agriculture  because  of  their 
relation  to  the  element  nitrogen,  this  being  commonly  considered  the 
most  valuable  element  of  plant  food.*  These  two  classes  of  bacteria 
are,  first,  the  nitrifying  bacteria,  and,  second,  the  nitrogen-gathering 
bacteria. 

THE  NITRIFYING  BACTERIA. 

The  nitrifying  bacteria  are  those  which  have  the  power  to  form 
nitrates.  In  the  following  brief  discussion  of  this  subject  we  include  at 
least  three  species  of  bacteria  which  by  their  combined  or  successive 
action  have  the  power  to  transform  organic  nitrogen  into  nitrate  nitro- 
gen, which  is  a  suitable  form  of  nitrogen  for  plant  food.  For  the  exact 
information  which  we  now  have  regarding  the  nitrifying  bacteria  we  are 
indebted  to  the  researches  of  Pasteur  and  Schlosing  and  Miintz  of 
France,  Winogradsky  of  Russia,  Warington  of  England,  and  others. 

The  nitrogen  in  the  soil  is  almost  entirely  in  organic  compounds;  that 
is,  the  nitrogen  (which  is  a  gas  in  the  free,  or  uncombined,  state)  is 
united  or  combined  with  other  elements,  notably  with  carbon,  hydrogen, 
and  oxygen,  in  the  form  of  partially  decayed  vegetable  or  organic  matter. 
(By  organic  matter  we  mean  matter  which  has  been  formed  by  the  growth 
of  some  organism,  either  plant  or  animal,  as  grass  or  flesh.)  Plants 
cannot  use  the  free  nitrogen  of  the  air  as  plant  food,  neither  can  they 
use  the  organic  compounds  of  nitrogen  which  occur  in  the  soil.  There 


*It  should  be  remembered  that  there  are  ten  essential  elements  of  plant  food 
each  of  which  is  of  equal  importance  to  the  plant,  for  if  the  plant  is  deprived  of  any 
one  of  the  ten  essential  elements  it  is  impossible  for  it  to  develop  and  mature.  Car- 
bon has  no  market  value  as  plant  food  because  the  plant  obtains  carbon  in  the  form  of 
of  carbon  dioxid,  a  gas  which  is  present  everywhere  in  the  atmosphere  and  which 
the  plant  inhales  through  its  leaves.  Both  hydrogen  and  oxygen  are  without 
market  value  because  they  are  the  elements  which  compose  water,  a  liquid  com- 
pound which  plants  absorb  through  their  roots.  Calcium,  magnesium,  iron,  and 
sulfur  have  no  market  value  as  elements  of  plant  food  because  they  are  present 
in  practically  all  soils  in  abundance  as  compared  with  the  amounts  required  in 
plant  growth.  The  three  elements  nitrogen,  phosphorus,  and  potassium,  do  have 
market  values,  because  they  are  required  by  plants  in  very  considerable  quantities, 
and  they  are  present  in  most  soils  in  rather  limited  amounts,  and  when  the  avail- 
able supply  of  any  one  of  these  elements  becomes  too  much  reduced  in  a  soil  the 
crop  yield  also  becomes  reduced.  For  further  information  regarding  the  use  of  these 
elements  of  plant  food  on  Illinois  soils,  see  Circular  No,  68,  "Methods  of  Maintain- 
ing the  Productive  Capacity  of  Illinois  Soils." 

307 


308  BULLETIN  No.  94.  [February, 

are  at  least  three  different  kinds  of  bacteria,  and  also  three  different 
steps  or  stages  involved  in  the  process  of  nitrification,  the  nitrogen  being 
changed  from  the  organic  compounds  first  into  the  ammonia*  form,  second, 
into  the  nitrite  form,  and  third  into  the  nitrate  form.  During  the 
process  the  nitrogen  is  separated  from  the  carbon  and  other  elements 
composing  the  insoluble  organic  matter,  and  is  united  or  combined  with 
oxygen  and  some  alkaline  element  (as  calcium)  to  form  the  soluble 
nitrate,  such  as  calcium  nitrate,  which  is  one  of  the  most  suitable  com- 
pounds of  nitrogen  for  plant  food.  Calcium  is  the  alkaline  element  con- 
tained in  lime  or  limestone.  The  name  calcium  nitrate  indicates  just 
what  elements  this  compound  contains;  namely,  calcium,  nitrogen,  and 
oxygen.  (In  the  names  of  compounds  the  ending  -ate  always  means 
oxygen.) 

This  is  the  general  process  of  nitrification  in  which  the  nitrifying 
bacteria  transform  or  transfer  the  nitrogen  from  insoluble  organic  com- 
pounds into  soluble  compounds  in  which  it  may  serve  as  available  plant 
food.  The  nitrate  which  is  thus  formed  may  be  calcium  nitrate  or 
magnesium  nitrate  or  potassium  nitrate  or  even  sodium  nitrate,  depend- 
ing upon  which  of  these  alkaline  elements  is  present  in  the  must  suitable 
form.  If  no  alkaline  element  is  present  in  available  form  then  no  nitrates 
can  be  made  in  the  soil.  One  of  the  reasons  for  applying  ground  lime- 
stone to  soils  which  are  deficient  in  lime  is  to  furnish  the  element 
calcium  in  suitable  form  for  the  formation  of  nitrates  in  the  process 
of  nitrification.  Ground  limestone  is  calcium  carbonate  (CaCO3),  a 
compound  containing  one  atom  of  calcium  (Ca),  one  atom  of  carbon  (C) 
and  three  atoms  of  oxygen  (03).  This  is  the  same  form  of  lime  which 
is  contained  naturally  in  limestone  soils — soils  which  are  noted  for  their 
great  productiveness — and  it  is  generally  the  most  economical  form  of 
lime  to  use  for  correcting  soil  acidity  and  promoting  nitrification. 

In  the  process  of  nitrification,  that  is  in  the  formation  of  nitrates, 
there  is  required,  not  only  the  presence  of  calcium,  or  some  other  alkaline 
element,  in  suitable  form,  but  also  a  good  supply  of  the  element  oxygen ; 
for  calcium  nitrate,  Ca(NO3)2,  contains  one  atom  of  calcium  (Ca),  two 
atoms  of  nitrogen  (N)2,  and  six  atoms  of  oxygen  (03)2,  in  each  molecule 
as  indicated  in  the  formula,  Ca(N03)2.  Magnesium  nitrate,  Mg(NO3)2, 
potassium  nitrate,  KNO3  (K  is  from  the  Latin  word  Kalium,  which  means 
potassium),  and  all  other  nitrates,  also,  contain  oxygen.  The  supply 
of  oxygen  for  the  formation  of  nitrates  in  the  soil  comes  from  the  air, 
which  consists  of  about  twenty  percent  oxygen,  seventy-eight  percent 
nitrogen,  and  two  percent  of  other  elements  and  compounds,  as  argon, 
carbon  dioxid,  COa,  water  vapor,  H2O,  etc.  One  of  the  important 
effects  of  cultivation,  or  tillage,  is  that  it  permits  the  air  more  freely  to 
enter  the  soil,  and  thus  promotes  nitrification. 

*  Technically  this  first  step  is  preliminary  to,  and  not  a  part  of,  nitrification. 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  309 

THE  NITROGEN-GATHERING  BACTERIA. 

As  stated  above,  the  nitrogen  naturally  in  the  soil  is  contained  almost 
entirely  in  the  organic  matter.  Any  process  which  tends  to  decompose 
or  destroy  this  organic  matter,  such  as  nitrification  or  other  forms  of 
oxidation,  will  also  tend  to  reduce  the  total  stock  of  nitrogen  in  the 
soil.  Because  of  this  fact  the  matter  of  restoring  nitrogen  to  the  soil 
becomes  of  very  great  importance.  Of  course  a  part  of  the  nitrogen 
removed  in  crops  may  be  returned  in  the  manure  produced  on  the  farm  ; 
and  nitrogen  may  also  be  bought  in  the  markets  in  such  forms  as  sodium 
nitrate  (containing'  15  to  16  percent  of  nitrogen),  ammonium  sulfate 
(containing  20  to  21  percent  of  nitrogen),  and  dried  blood  (containing 
12  to  15  percent  nitrogen) ;  but,  when  we  bear  in  mind  that  such  com- 
mercial nitrogen  costs  about  15  cents  a  pound,  and  that  one  bushel  of 
corn  contains  about  one  pound  of  nitrogen,  it  will  be  seen  at  once  that 
the  purchase  of  nitrogen  cannot  be  considered  practicable  in  general 
farming,  although  in  market  gardening,  and  in  some  other  kinds  of 
intensive  agriculture,  commercial  nitrogen  can  often  be  used  with  very 
marked  profit. 

Nitrogen  is  removed  from  the  soil  not  only  in  the  crops  grown,  but 
also,  and  frequently  in  larger  amounts  per  annum,  in  the  drainage 
waters,  and  in  some  other  ways,  as  by  denitrification  and  by  the  blow- 
ing and  washing  of  the  surface  soil.  Professor  Snyder,  of  the  Minnesota 
Experiment  Station,  has  shown  that  during  a  series  of  years  the  total 
loss  of  nitrogen  from  some  Minnesota  soils  in  some  cases  amounts  to 
several  times  the  amount  actually  used  in  the  crops  produced. 

Considering  all  of  these  facts,  and  the  additional  facts  that  there  are 
about  seventy-five  million  pounds  of  atmospheric  nitrogen  resting  upon 
every  acre  of  land,  and  that  it  is  possible  to  obtain  unlimited  quantities 
of  nitrogen  from  the  air  for  use  of  farm  crops,  and  at  very  small  cost,  the 
inevitable  conclusion  is  that  the  inexhaustible  supply  of  nitrogen  in  the 
air  is  the  store  from  which  we  must  draw  to  maintain  a  sufficient  amount 
of  this  element  in  the  soil  for  the  most  profitable  crop  yields. 

It  is  often  stated  that  leguminous  plants,  such  as  clover,  have  power 
to  obtain  free  nitrogen  from  the  air.  This  is  not  strictly  true.  Red 
clover,  for  example,  has  no  power  in  itself  to  get  nitrogen  from  the  air. 
It  is  true,  however,  that  the  microscopic  organisms*  which  commonly 
live  in  tubercles  upon  the  roots  of  the  clover  plant  do  have  the  power  to 
take  free  nitrogen  from  the  air  and  cause  it  to  unite  with  other  elements 
to  form  compounds  suitable  for  plant  food.  The  clover  plant  then  draws 


*Among  the  scientists  who  were  prominent  in  making  these  discoveries  regard- 
ing the  action  of  bacteria  in  the  fixation  of  atmospheric  nitrogen  were  Hellriegel, 
Willfarth,  and  Nobbe  in  Germany,  Atwater  in  America,  Lawes  and  Gilbert  in  Eng- 
land, and  Boussingault  and  Ville  in  France. 


310  BULLETIN  No.  94.  [February, 

upon  this  combined  nitrogen  in  the  root  tubercles,  and  makes  use  of  it 
in  its  own  growth,  both  in  the  tops  and  in  the  roots  of  the  plant. 

These  nitrogen-gathering  bacteria  live  in  tubercles  upon  the  roots 
of  various  leguminous  plants,*  such  as  red  clover,  white  clover,  alfalfa, 
sweet  clover,  cowpeas,  soy  beans,  vetch,  field-peas,  garden-peas,  field 
and  garden  beans,  etc.  These  tubercles  vary  in  size  from  a  pinhead  to 
a  pea,  varying  with  the  different  kinds  of  plants,  being  especially  small 
upon  some  of  the  clovers,  and  very  large  upon  cowpeas  and  soy  beans. 


*It  may  be  well  to  call  attention  to  the  fact  that  there  are  numerous  instances 
where  two  different  kinds  of  plants  live  together  in  intimate  partnership  relation. 
If  only  one  of  the  two  plants  receives  benefit  from  this  relationship  or  association, 
then  the  plant  receiving  the  benefit  is  called  a  parasite.  Thus  the  mistletoe  is  a 
parasite  upon  the  elm  or  gum  or  other  tree  on  which  it  lives.  The  mistletoe  draws 
its  nourishment  from  the  tree.  The  tree  is  injured  rather  than  benefited  by  the 
mistletoe.  Dodder  is  also  a  parasitic  plant,  living  upon  other  plants,  except  during 
the  early  part  of  its  growth.  Ticks  and  lice  are  common  examples  of  animal  para- 
sites living  upon  other  animals. 

In  some  cases  a  relationship  exists  which  is  not  parasitic  but  symbiotic.  The 
term  symbiosis,  which  is  commonly  used  by  biologists  to  define  this  relationship, 
means  living  together  in  mutual  helpfulness.  The  association  of  bees  and  flowers 
may  serve  to  illustrate  this  mutual  helpfulness,  although  this  is  not  an  example  of 
intimate  symbiosis.  Thus  the  bees  obtain  their  food  from  the  flowers  and,  in  turn, 
the  flowers,  many  of  them,  are  incapable  of  producing  seed  or  fruit  unless  the  pollen 
is  carried  from  the  male  flower  to  the  female  flower  by  bees  or  other  agencies.  It  is 
well  known  that  plant  lice  and  ants  are  mutually  helpful. 

Likewise  the  association  of  nitrogen-gathering  bacteria  and  leguminous  plants  is 
a  relationship  of  mutual  helpfulness  and  this  is  one  of  the  best  illustrations  of  what 
is  meant  by  symbiosis.  The  legume  furnishes  a  home  for  the  bacteria  and  also 
furnishes  in  its  juice  or  sap  most  of  the  nourishment  upon  which  the  bacteria  live. 
The  bacteria,  on  the  other  hand,  take  nitrogen  from  the  air  contained  in  the  pores  of 
the  soil,  and  cause  this  nitrogen  to  combine  with  other  elements  in  suitable  form 
for  plant  food  which  is  then  given  up  to  the  legume  for  its  own  nourishment. 

Another  illustration  of  remarkable  parasitism,  if  not,  indeed,  one  of  true  sym- 
biosis, is  found  in  the  common  lichens  living  upon  rocks  and  trees.  The  lichen  is 
not  a  single  plant,  but  two  plants — an  alga,  which  lives  upon  the  wood  or  stone, 
and  a  fungus  which  lives  upon  the  alga.  Algae  also  live  in  the  free  state  separate 
from  fungi,  and  the  present  opinion  of  botanists  seems  to  be  that  when  the  two  are 
associated  in  the  form  of  lichens  this  association  is  not  detrimental,  but  rather 
beneficial,  to  the  alga,  as  well  as  to  the  parasitic  fungus.  If  this  is  true,  then  it  is 
another  case  of  true  symbiosis.  (There  is  reason  to  believe  that  the  fungus  has 
some  power  to  feed  upon  atmospheric  nitrogen,  and  then  to  furnish  combined 
nitrogen  to  the  alga  upon  which  it  lives.) 

In  the  symbiosis  of  leguminous  plants  and  nitrogen-gathering  bacteria  we  have 
a  partnership  or  relationship  of  immeasurable  value  to  agriculture.  Here  is  a  class 
of  plants  (legumes)  that  are  capable  of  consuming  or  utilizing  nitrogen  in  quantities 
larger  than  could  possibly  be  obtained  from  ordinary  soils  for  any  considerable 
length  of  time.  They  have  no  power  in  themselves  of  taking  nitrogen  from  the 
atmosphere,  and  to  them  the  symbiotic  relation  with  this  low  order  of  plants  (the 
nitrogen-gathering  bacteria)  is  especially  helpful,  and  for  the  best  results  it  is 
absolutely  necessary. 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  311 

The  tubercles  are,  of  course,  easily  seen  with  the  eye,  but  the  tubercle  is 
only  the  home  of  the  bacteria,  somewhat  as  the  ball  upon  the  willow 
twig  is  the  home  of  the  insects  within.  The  bacteria  themselves  are  far 
too  small  to  be  seen  with  the  unaided  eye,  although  they  can  be  seen  by 
means  of  the  most  powerful  microscope.  Several  million  bacteria  may 
inhabit  a  single  tubercle.  It  is  not  necessary  to  see  the  bacteria,  because 
if  we  find  the  tubercles  upon  the  roots  of  the  plant,  we  know  that  the 
bacteria  are  present  within,  as  otherwise  the  tubercle  would  not  be 
formed. 

Although  the  plant  itself,  as  clover,  for  example,  has  no  power  to 
feed  upon  the  free  or  uncombined  nitrogen  in  the  air,  yet  these  nitrogen- 
gathering  bacteria  do  have  the  power  to  absorb  the  free  nitrogen  and 
cause  it  to  combine  with  other  elements,  forming  nitrates  or  other  com- 
pounds wnich  are  suitable  forms  of  nitrogen  for  plant  food. 

It  has  also  been  demonstrated  that,  as  a  rule,  there  are  different 
species  of  nitrogen-gathering  bacteria  for  markedly  different  species  of 
leguminous  plants.  Thus  we  have  one  kind  of  bacteria  for  red  clover, 
another  kind  for  cowpeas,  another  kind  for  soy  beans,  and  still  a  different 
kind  for  alfalfa.* 

THE  RED  CLOVER  BACTERIA. 

That  clover  has  no  power  in  itself  to  gather  atmospheric  nitrogen, 
and  that  the  bacteria  do  have  power  to  feed  the  clover  plant  with  nitrogen 
gathered  from  the  air  is  very  easy  to  demonstrate.  It  is  one  of  the 
regular  laboratory  practices  of  the  students  in  soil  fertility  in  the  Agri- 
cultural College  to  make  this  demonstration.  Plate  1  is  an  illustration 
of  such  student  work.  The  two  pots  which  are  shown  were  provided 
with  all  elements  of  plant  food,  excepting  the  one  element  nitrogen. 
Thus  far  the  two  pots  are  exactly  alike.  Each  contains  no  nitrogen,  as 
indicated  by  the  label  "No  N."  Each  pot  is  planted  with  the  same 
number  of  red  clover  seeds.  To  the  right-hand  pot,  however,  some 
bacteria  ("Bac.")  were  added,  while  none  were  added  to  the  left-hand 
pot.  These  bacteria  were  obtained  by  taking  about  one  pound  of  soil 
from  a  clover-field  where  abundance  of  tubercles  were  found  on  the 


*There  are  some  noteworthy  exceptions  to  this  rule  (see  following  pages  for 
illustration),  and  there  is  some  evidence  that,  by  a  comparatively  long  process  of 
breeding,  or  evolution,  the  bacteria  which  naturally  live  upon  one  kind  of  legume 
may  gradually  develop  the  power  to  live  upon  a  distinctly  different  legume  to  which 
they  were  not  at  first  adapted.  Of  course  this  process  of  forcing  bacteria  to  live 
upon  a  legume  to  which  they  are  not  naturally  adapted  has  little  or  no  practical 
value  because  it  is  unnecessary  if  there  is  a  species  of  bacteria  which  naturally 
lives  upon  the  same  legume.  On  the  other  hand,  if,  by  any  such  process  of  breed- 
ing, or  evolution,  a  species  of  nitrogen-gathering  bacteria  could  be  developed  which 
could  live  on  a  non-leguminous  plant,  as  corn,  for  example,  it  would  be  of  incal- 
culable practical  value.  As  yet  the  efforts  of  bacteriologists,  working  on  this  prob- 
lem, have  given  only  negative  results,  so  far  as  known  to  the  writer. 


312 


BULLETIN  No.  94. 


[February, 


PLATE  1.     RED  CLOVER:  EFFECT  OF  BACTERIA.     No  NITROGEN  IN  THE 
SOIL  OF  EITHER  POT. 

clover  roots,  adding  this  soil  to  about  one  quart  of  pure  water,  shaking 
for  a  few  minutes,  allowing  the  soil  to  settle,  then  taking  a  small  quan- 
tity of  almost  clear  solution,  and  adding  it  to  the  pot  which  we  wished 
to  inoculate  with  the  red  clover  bacteria.  Aside  from  the  addition  of 
these  microscopic  bacteria  to  the  right-hand  pot,  these  two  pots  were 
treated  exactly  alike  throughout  the  experiment.  It  will  be  plainly 
seen  that  where  the  bacteria  were  added  the  clover  was  furnished  with 
sufficient  nitrogen  to  make  a  strong  and  luxuriant  growth,  while  without 
the  bacteria  the  clover  (in  the  left-hand  pot)  only  germinated  and  made 
what  little  growth  it  could  with  the  small  amount  of  nitrogen  contained 
in  the  seed.  This  result  is  the  difference  between  success  and  failure 
of  the  clover  crop. 

In  general  the  clover  bacteria  are  well  distributed  over  the  northern 
and  central  part  of  Illinois,  but  we  now  have  some  very  strong  evidence 
that  they  are  not  well  distributed  in  some  soils  of  large  area  in  southern 
Illinois.  There  is  also  some  evidence  that  they  were  not  originally 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  313 

present  even  in  the  soils  where  they  are  now  found  in  great  abundance; 
and,  furthermore,  it  seems  very  probable  that  these  bacteria  may  cease 
to  live  in  a  soil  where  they  have  once  been  present,  provided  clover  is 
not  grown  on  the  land  for  several  years. 

It  will  help  us  to  understand  this  matter  if  we  bear  in  mind  that  the 
home  of  these  bacteria  is  the  tubercle  upon  the  clover  root.  It  is  quite 
evident  that  they  will  continue  to  live  upon  the  decaying  tubercles  or 
roots  for  three  or  four  years  after  the  clover  plant  has  been  killed.  On 
the  other  hand,  we  have  some  notable  evidence  that  the  bacteria  do 
not  continue  to  live  in  a  soil  after  five  or  six  years'  continuous  cropping 
with  absolutely  no  clover  growing  on  the  land  during  those  years.  It 
is  a  simple  matter  for  any  one  to  determine  whether  the  bacteria  are 
present  or  not,  for  the  tubercles  which  are  formed  if  the  bacteria  are 
present  are  plainly  seen  attached  to  small  roots.  They  look  somewhat 
like  miniature  potatoes,  varying  in  size  from  pinheads  on  clover  to  peas 
on  soy  beans  or  cowpeas.  (See  Plates  2  and  4.)  It  is  important  to 
remember  that  the  bacteria  live  in  the  soil  and  not  in  the  seed. 

When  clover  is  cut  for  seed,  it  is  frequently  left  to  lie  upon  the  ground 
until  the  straw  becomes  half  rotten  and  very  dirty;  and,  consequently 
when  it  is  threshed,  it  practically  always  happens  that  there  is  at  least 
some  small  amount  of  dust  and  dirt  taken  with  the  seed.  This  dirt  is 
almost  sure  to  cany  with  it  some  bacteria  from  the  soil.  If  these  few 
bacteria  are  scattered  with  the  clover  seed  when  it  is  sowed  they  will 
inoculate  at  least  a  few  plants,  and  if  they  are  allowed  to  multiply  on 
these  plants,  and  especially  if  the  same  field  is  repeatedly  seeded  with 
clover,  the  soil  will  ultimately  become  thoroughly  infected  with  the 
clover  bacteria.  Of  course  they  may  be  carried  from  one  part  of  the 
farm  to  another,  or  even  from  one  farm  to  another,  by  various  agencies, 
as  dust  or  wind  storms,  surface  drainage  or  flood  waters,  manure  made 
from  clover  hay.  implements  used  in  cultivating  the  soil,  etc.,  etc. 

Many  of  the  older  farmers  of  Illinois  have  stated  to  the  writer  that 
when  this  country  was  very  new  it  was  commonly  found  difficult  to  get 
a  "catch"  of  clover  on  new  land.  After  a  good  "catch"  was  once  gotten, 
then  it  was  easier  to  get  clover  to  grow  on  that  land  the  next  time.  There 
was  a  saying  among  the  farmers  that  clover  would  not  do  well  until  they 
got  the  "wild  nature"  out  of  the  land.  Their  final  success  was  undoubt- 
edly due,  not  to  getting  anything  out  of  the  land,  but  rather  to  getting  the 
bacteria  into  the  land.  Several  Illinois  farmers  have  reported  some 
quite  remarkable  results  from  very  light  applications  of  the  clover  chaff 
or  straw  (obtained  in  hulling  clover)  in  its  beneficial  effect  on  clover  on 
land  where  it  was  otherwise  difficult  to  get  a  "catch."  There  is  a  some- 
what general  belief  among  farmers  of  long  experience  that  clover  straw 
or  chaff  has  some  special  value  in  getting  a  catch  of  clover  aside  from  its 
value  as  manure  or  for  the  seed  which  it  sometimes  contains. 


314  BULLETIN  No.  94.  [February, 

Manager  F.  A.  Warner  of  the  Sibley  Estate,  Ford  County,  recently 
stated  to  the  writer  that  they  had  had  very  great  difficulty  to  get  clover 
to  grow  when  they  first  began  growing  clover  on  that  large  estate,  some 
six  or  eight  years  ago,  although,  after  a  good  crop  was  once  secured,  they 
rarely  had  any  further  difficulty  in  getting  a  catch  of  clover  on  the  same 
land. 

On  the  common  gray  prairie  soil  of  the  Lower  Illinoisan  Glaciation,  in 
southern  Illinois,  the  commonest  type  of  soil  in  more  than  twenty  coun- 
ties, practically  no  red  clover  is  grown.  In  the  spring  of  1903  we  seeded 
red  clover  on  that  type  of  soil  in  three  places ;  namely,  on  the  University 
of  Illinois  soil  experiment  fields  near  Edgewood,  Effingham  County,  near 
Du  Bois,  Washington  County,  and  near  Cutler,  Perry  County.  On  certain 
plots  the  soil  acidity  had  been  corrected  with  lime  and  an  abundant  sup- 
ply of  phosphorus  (in  bone  meal)  had  been  provided,  potassium  also 
having  been  added  on  some  plots.  These  fields  were  carefully  examined 
the  latter  part  of  June,  and  at  Cutler  and  Du  Bois  the  clover  was  found 
to  be  dead  or  dying,  and  no  tubercles  could  be  found  upon  the  clover 
roots,  although  on  the  clover  which  had  been  seeded  at  about  the  same 
time  on  the  University  fields  at  Urbana  the  root  tubercles  were  found 
in  great  abundance.  At  Edgewood  a  few  tubercles  were  found  and  the 
clover  appeared  to  be  growing  fairly  well.  Infected  red  clover  soil  was 
at  once  procured  and  scattered  over  the  fields  at  Edgewood,  Du  Bois,  and 
Cutler,  but  it  was  evidently  too  late  to  be  of  any  marked  benefit.  At 
Cutler  and  at  Du  Bois  the  clover  was  a  complete  failure.  (It  will  be  tried 
again  next  year.)  At  Edgewood  it  continued  to  grow  fairly  well,  and 
its  progress  next  season  (1904)  will  be  watched  with  much  interest.  It 
should  be  stated  that  the  Experiment  Station  has  been  growing  clover 
for  several  years  with  varying  degrees  of  failure  on  land  adjoining  the 
present  clover  field  at  Edgewood,  and  it  is  possible  that  this  year's 
apparent  success  from  the  start  is  due  in  part  at  least  to  the  bacteria 
which  have  been  incidentally  introduced  and  multiplied  year  after  year 
and  scattered  over  the  adjoining  land  by  wind  and  dust  storms.  Before 
the  close  of  the  season  the  tubercles  developed  in  abundance  on  the  roots 
of  the  clover  at  Edgewood. 

An  experience  reported  by  Professor  Herbert  W.  Mumford,  of  the 
Animal  Husbandry  Department  of  this  university,  will  be  of  interest 
and  value  in  this  connection.  Professor  Mumford  commonly  grows 
clover  in  his  rotations  on  his  own  private  farm,  but  he  states  that  at  one 
time  one  particular  field  was  cropped  continuously  with  timothy,  oats, 
and  corn  for  some  six  years  or  more  without  any  clover  whatever.  It 
was  then  again  seeded  to  clover,  but  the  crop  made  a  complete  failure, 
although  on  other  land  where  clover  had  been  grown  more  recently  a  suc- 
cessful clover  crop  was  grown  from  the  same  kind  of  seed  seeded  at  about 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  315 

the  same  time.  The  following  year  this  particular  field  was  again  seeded 
to  clover.  This  time  the  "catch"  was  not  a  total  failure,  but  it  was  too 
poor  to  save,  and  it  was  plowed  up  and  the  land  again  seeded  to  clover 
the  next  year,  and  an  excellent  catch  of  clover  resulted.  After  this, 
clover  was  frequently  grown  on  this  field,  and  no  special  difficulty  was 
had  in  getting  good  crops. 

While  the  failure  of  clover  may  often  be  due  to  drouth,  and  in  some 
places  due  to  soil  acidity  (lack  of  lime),  and  sometimes  even  due  to  an 
insufficient  supply  of  available  phosphorus  or  of  potassium,  we  now 
know  with  certainty  that  it  sometimes  fails  because  of  the  absence  of 
the  nitrogen-gathering  bacteria,  especially  on  land  which  has  never  grown 
clover,  and  probably  also  on  land  which  has  not  grown  it  recently.  We 
should  always  remember  that  the  bacteria  do  not  thrive  in  strongly  acid 
soils.  Even  though  they  may  sometimes  live  in  such  soils  and  perhaps 
produce  some  tubercles  upon  the  roots  of  certain  hardy,  strong  growing 
legumes,  like  cowpeas,  nevertheless  we  are  obtaining  some  strong  evi- 
dence that  in  such  acid  soils  they  have  but  little  power  to  gather  nitro- 
gen from  the  air.  That  ground  limestone  is  the  most  economical  and 
satisfactory  material  to  use  in  correcting  the  acidity  of  soils  is  strongly 
indicated  by  the  information  we  have  thus  far  obtained.  On  the 
upland  prairie  soils  of  the  Lower  Illinoisan  Glaciation  where  red  clover 
has  never  been  grown  successfully,  largely  because  of  the  acidity  of  the 
soil,  it  will  undoubtedly  be  helpful  and  profitable  not  only  to  correct  the 
acidity  of  the  soil  with  ground  limestone,  but  also  to  secure  infected 
soil  from  some  field  of  timber  land  or  bottom  land  where  red  clover  is 
growing,  well  provided  with  root  tubercles,  and  inoculate  the  field 
with  it.  This  soil  should  be  collected  to  a  depth  of  three  or  four  inches 
and  scattered  over  the  prairie  land  at  the  rate  of  a  few  hundred  pounds 
per  acre  at  the  time  the  clover  is  seeded  or  before. 

THE  COWPEA  BACTERIA. 

Plate  2  is  made  from  a  photograph  of  a  cowpea  root  with  the  tubercles 
upon  it.  This  illustration  shows  the  cpwpea  tubercles  at  nearly  natural 
size,  which  is  about  as  large  as  the  seed  ef  ordinary  garden  peas. 

The  cowpea  bacteria  are  already  quite  widely  distributed  in  southern 
Illinois,  especially  where  this  crop' has  been  grown  for  several  years,  but 
they  are  not  common  in  the  soils  of  other  parts  of  the  state.  It  is  doubt- 
ful, however,  if  it  is  necessary  or  even  worth  while  to  take  the  trouble  to 
inoculate  soil  for  cowpeas.  Some  few  tubercles  almost  invariably 
develop  on  cowpea  roots  the  first  year  they  are  seeded,  even  where  they 
have  never  been  grown  before,  and  if  seeded  the  second  year  on  the  same 
land  the  plants  are  usually  abundantly  provided  with  root  tubercles. 


316 


BULLETIN  No.  94. 


[February, 


PLATE  2.     COWPEA  ROOT  TUBERCLES,  NATURAL  SIZE. 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  317 

Just  why  the  cowpea  bacteria  develop  so  rapidly  even  without  special 
inoculation  is  not  definitely  known.  It  may  be  that  the  same  bacteria 
also  live  on  some  other  leguminous  plant  which  is  more  or  less  widely 
distributed  over  the  state,  but  it  seems  more  likely  that  the  bacteria 
are  brought  with  the  seed.  As  a  matter  of  fact,  the  cowpea  harvest  is 
usually  dirty.  This  is  an  annual  plant,  and  consequently  the  crop  is 
grown  on  recently  plowed  land  and  is  sometimes  cultivated  during  the 
season.  Cowpeas  are  commonly  harvested  with  a  mowing  machine  and 
then  raked  up  on  the  loose  ground.  When  they  are  threshed  more  or 
less  dirt  remains  with  the  seed.  Furthermore,  the  seed  coats  are  not 
infrequently  cracked,  thus  providing  an  excellent  place  for  the  lodg- 
ment of  particles  of  soil. 

Whether  it  would  be  profitable  to  inoculate  the  land  for  cowpeas 
would  depend  very  largely  upon  the  difficulty  or  cost  of  obtaining 
the  infected  material.  If  soil  thoroughly  infected  with  the  cowpea 
bacteria  can  be  scattered  over  the  land  at  the  rate  of  about  2,000  pounds 
to  the  acre  at  a  cost  of  $1.00  or  less  per  ton,  it  might  prove  profitable. 
It  is  doubtful  if  a  light  application  of  100  or  200  pounds  would  produce 
any  very  marked  effect  in  the  yield  the  first  season.  After  the  soil 
becomes  well  infected  the  cowpeas  then  obtain  much  nitrogen  from  the  air, 
and  the  yield  of  cowpeas  is  likely  to  be  largely  increased.  Of  course 
there  is  no  fixation  of  atmospheric  nitrogen  if  there  are  no  tubercles  on 
the  roots. 

In  1902  several  plots  of  cowpeas  were  seeded  on  the  soil  experiment 
field  at  the  university.  One  of  these  plots  (404)  had  become  thoroughly 
infected  with  the  cowpea  bacteria  because  of  its  being  so  situated  that 
more  or  less  surface  drainage  water  flowed  over  it  from  an  adjacent  field 
upon  which  cowpeas  had  been  grown  for  three  successive  years.  Another 
plot  (408),  owing  to  a  slightly  different  situation,  had  not  become  infected. 
The  two  plots  were  seeded  in  July  after  a  crop  of  oats  had  been  removed 
from  the  land.  Within  three  weeks  after  seeding,  numerous  root  tubercles 
could  be  found  on  the  plants  on  the  infected  plot.  Later  on,  ten  average 
consecutive  plants  were  taken  up  as  completely  as  possible,  and  412 
tubercles  were  found  on  the  roots,  making  an  average  of  more  than  40 
tubercles  to  the  plant.  On  Plot  408  only  an  occasional  plant  was  found 
infected,  and  such  plants  would  usually  have  only  a  single  large  tubercle 
on  their  roots.  Ten  average  plants  not  infected  were  collected  from 
Plot  408  for  comparison  with  the  ten  infected  plants  from  Plot  404. 


318 


BULLETIN  No.  94. 


[February, 


PLATE  3.     COWPEAS:  EFFECT  OF  BACTERIA  IN  ORDINARY  ILLINOIS  BLACK 

PRAIRIE  SOIL. 

Plate  3  shows  these  two  bunches  of  plants,  the  infected  plants  with 
root  tubercles  on  the  right,  and  the  plants  without  tubercles  on  the  left. 
Four  more  sets  of  ten  plants  each  were  then  collected,  two  sets  from 
Plot  404  and  two  from  Plot  408.  Each  set  of  infected  plants  was  sepa- 
rated into  three  parts,  (1)  tops,  (2)  roots,  (3)  tubercles;  and  each  set  of 
plants  not  infected  was  separated  into  (1)  tops,  and  (2)  roots.  All  of 
these  samples  were  dried  and  analyzed  for  nitrogen.  The  results  obtained 
are  shown  in  Table  I. 

The  results  clearly  show  the  very  great  value  of  the  nitrogen- 
gathering  bacteria  in  growing  cowpeas.  In  each  of  the  separate  trials 
A,  B,  and  C,  the  infected  plants  contained  about  twice  as  much  total 
dry  matter  as  the  plants  not  infected.  The  infected  plants  also  contained 
a  much  higher  percent  of  nitrogen  than  the  plants  not  infected,  the 
infected  plants  containing  4.09  to  4.33  percent  in  the  tops  and  1.45  to 
1.53  percent  in  the  roots,  while  those  not  infected  contained  only  2.32 


1904.] 


NITROGEN  BACTERIA  AND  LEGUMES. 


319 


TABLE  I. — -FIXATION  OF  NITROGEN  BY  COWPEAS. 


COWPEA  PLANTS. 

Dry 
matter, 
cgs. 

Nitrogen, 
content, 
percent. 

Nitrogen, 
amount, 
cgs. 

Nitrogen  fixed 
by  bacteria, 
cgs. 

No. 

Part  . 

Al  —  Ten  plants, 
with  bacteria 
present  

Tops  . 

3580 
620 
190 

4  09 
1.45 
5.97 

146 
9 
II 

125 

Roots  

Tubercles  .... 

A2  —  Ten   plants, 
without    bacteria 

Total  

4390 

166 

Tops  . 

1560 
300 

2.42 
.88 

38 
3 

Roots  

Total  

1860 

41 

HI—  Ten  plants, 
with  bacteria 
present  

Tops  . 

3970 
690 
300 

4.31 
1.47 
6.05 

171 
10 
18 

140 

Roots  ... 

Tubercles 

B2—  Ten  plants, 
without   bacteria 

Total  
Tops  . 

4960 

199 

2060 
430 

2.69 

.88 

55 
4 

Roots  

Total  

2J90 

59 

Cl—  -Ten   plants, 
with   bacteria 
present  

Tops  . 

3300 
520 
290 

4.33 
1.53 
5.76 

143 
8 
17 

124 

Roots  

Tubercles 

C2  —  Ten   plants, 
without    bacteria 

Total  .  .  . 

4110 

168 

Tops  . 

1730 
400 

2.32 
.88 

40 
4 

Roots  

Total  

2130 

44 

to  2.69  percent  in  the  tops  and  .88  percent  in  the  roots.  Besides  this, 
the  tubercles  on  the  infected  plants  contain  5.76  to  6.05  percent  of  nitro- 
gen. In  these  young  and  rapidly  growing  plants  the  tubercles  are  much 
richer  in  nitrogen  than  any  other  part  of  the  plant.  It  should  be  stated 
that  as  the  plants  approach  maturity  the  nitrogen  is  largely  absorbed 
from  the  tubercles  and  stored  in  the  tops  and  roots.  At  the  time  these 
plants  were  taken  up  the  tubercles  actually  contained  more  nitrogen  than 
the  roots.  The  infected  plants  contained  nearly  four  times  as  much 
nitrogen  as  the  plants  not  infected,  and  about  three-fourths  of  the  total 
nitrogen  in  the  infected  plants  was  obtained  from  the  air.  The  roots  and 
tubercles  of  the  infected  plants  contained  six  to  seven  times  as  much 
nitrogen  as  the  roots  of  the  plants  not  infected. 

THE  SOY  BEAN  BACTERIA. 

Soy  bean  bacteria  are  evidently  much  less  likely  to  be  carried  with 
the  seed  than  are  the  cowpea  bacteria.  The  soy  bean  plant  grows  more 
erectly  than  the  cowpea  (see  Circular  No.  69,  "The  Cowpea  and  Soy  Bean 
in  Illinois"),  and  the  crop  is  quite  commonly  harvested  with  a  self- 


320  BULLETIN  No.  94.  [February, 

binder  which  keeps  it  quite  free  from  dirt.  The  soy  bean  seed  is  nearly 
round  and  smooth,  and  the  seed  coat  is  not  commonly  cracked.  These 
facts  may  explain  why  the  soy  bean  seed  carry  so  few  bacteria  as  com- 
pared with  cowpeas. 

On  one  of  the  soil  experiment  fields  on  the  university  farm  at  Urbana, 
where  soy  beans  have  been  grown  for  three  years,  no  tubercles  could  be 
found  on  the  plants  either  the  first  or  second  year,  and  only  an  occasional 
plant  with  tubercles  could  be  found  the  third  year.  In  1902  a  series 
of  plots,  some  of  which  had  been  treated  in  different  ways  with  applica- 
tions of  limestone,  phosphorus,  and  potassium,  were  seeded  with  soy 
beans.  No  tubercles  could  be  found  at  any  time  during  the  season  on 
the  soy  beans  growing  on  any  of  the  different  plots.  In  1903  the  same 
plots  were  again  seeded  to  soy  beans,  and  at  the  same  time  part  of  each 
plot  was  inoculated  with  infected  soy  bean  soil  drilled  in  with  the  seed 
at  the  rate  of  about  500  pounds  of  infected  soil  to  the  acre.  When  the 
plants  were  only  a  few  weeks  old  tubercles  were  to  be  found  upon  many 
plants  growing  where  the  infected  soil  had  been  applied,  and  before  the 
close  of  the  season  at  least  half  of  these  plants  in  the  inoculated  part  of 
the  field  had  one  or  more  tubercles  upon  their  roots,  and  some  plants 
could  be  found  whose  roots  were  abundantly  provided  with  tubercles. 
(See  Plate  4.) 

On  the  uninoculated  part  of  the  field  soy  bean  plants  were  examined 
probably  fifty  times  during  the  season,  several  plants  being  taken  up 
each  time,  but  not  a  single  tubercle  was  found  at  any  time,  notwithstand- 
ing that  this  was  the  second  crop  of  soy  beans  upon  this  soil.  Of  course 
the  inoculated  part  of  the  field  did  not  become  sufficiently  infected  to 
markedly  benefit  the  1903  crop,  but  it  is  planned  to  grow  soy  beans  upon 
this  field  again  in  1904  when  the  bacteria  will  doubtless  have  multiplied 
sufficiently  to  produce  marked  results  in  the  growth  of  the  crop. 

From  these  and  from  other  somewhat  similar  experiments  it  is  con- 
cluded that  as  a  rule  soy  beans  should  be  inoculated  when  they  are  first 
seeded,  and  that  they  should  then  be  grown  a  second  year  upon  the  same 
land.  If  soy  beans  are  afterward  grown  upon  this  land  once  in  every 
three  or  four  years,  the  soil  will  doubtless  remain  well  infected  with  the 
soy  bean  bacteria. 

It  is  believed  that,  100  pounds  of  infected  soy  bean  soil  per  acre  will 
be  sufficient  to  produce  a  thorough  infection  the  second  year,  and  it  is 
improbable  that  one  ton  of  infected  soil  per  acre  would  produce  a  thor- 
ough inoculation  .the  first  season.  One  ton  is  only  twenty  times  100 
pounds,  while  one  tubercle  which  will  be  produced  during  a  single  season 
from  a  single  bacterium  may  contain  many  million  bacteria,  thus  it  will 
be  seen  that  it  will  be  more  economical  to  inoculate  rather  lightly  and 
allow  the  bacteria  to  multiply  themselves  rather  than  to  inoculate 
heavily  at  great  expense. 


1904.] 


NITROGEN  BACTERIA  AND  LEGUMES. 


321 


PLATE  4.     SOY  BEAN  ROOT  TUBERCLES,  NATURAL  SIZE. 


322  BULLETIN  No.  94.  [February, 

It  may  be  stated  that  the  infected  soy  bean  soil  used  in  these  experi- 
ments was  obtained  from  Mr.  A.  A.  Hinkley  of  Du  Bois,  Illinois,  who 
has  been  growing  soy  beans  on  the  same  land  for  many  years  until  it  has 
become  well  infected.  Mr.  Hinkley  has  consented  to  furnish  infected 
soy  bean  soil  so  far  as  he  is  able  to  do  without  serious  interference  with 
his  regular  work,  to  any  one  who  may  desire  it,  at  a  price  which  will 
cover  his  expense  and  loss.  This  will  probably  amount  to  about  $1.00 
for  the  first  100  pounds  and  fifty  cents  for  each  additional  100  pounds, 
in  the  shipment,  including  the  cost  of  bags,  the  purchaser  to  pay  freight 
from  Bois  station,  which  is  located  in  Washington  County,  Illinois,  on 
the  Illinois  Central  Railroad. 

THE  ALFALFA  AND  SWEET  CLOVER  BACTERIA. 

That  soil  inoculation  with  alfalfa  bacteria  is  commonly  of  very  great 
value  in  growing  alfalfa  has  been  shown  very  conclusively  by  the  investi- 
gations reported  in  Bulletin  No.  76,*  "Alfalfa  on  Illinois  Soil."  In  some 
places,  however,  inoculation  was  found  to  be  unnecessary.  A  careful 
and  extensive  investigation  of  alfalfa  growing  in  different  parts  of  Illinois 
revealed  the  fact,  as  stated  in  Bulletin  76,  "that  the  alfalfa  bacteria  are 
certainly  present  in  some  places  in  the  state  while  in  most  other  places 
they  are  certainly  not  present  in  sufficient  number  to  become  of  appre- 
ciable assistance  to  the  alfalfa  within  three  or  four  years,  and  the  question 
naturally  arises  how  it  happens  that  some  fields  are  already  infected 
while  others  are  not."  It  was  suggested  in  that  bulletin  that  the  alfalfa 
bacteria  may  "live  on  some  other  plants  besides  alfalfa  and  that  one  of 
these  plants  is  native  or  has  been  introduced  in  certain  sections"  of  the 
state.  It  was  also  suggested  "that  a  few  bacteria  are  always  carried  with 
alfalfa  seed,  and  that  if  the  alfalfa  is  grown  continuously  or  repeatedly 
in  any  place  the  soil  will  finally  become  thoroughly  infected,  and  the 
bacteria  will  then  be  carried  by  flood  waters,  dust  storms,  etc.,  over 
adjoining  fields,  and  possibly  for  long  distances,  especially  along  river 
valleys."  This  latter  suggestion  was  known  to  be  a  fact  at  the  time  it 
was  written;  and  subsequent  investigations  have  furnished  conclusive 
proof  that  the  alfalfa  bacteria  do  live  upon  another  plant;  namely,  the 
ordinary  sweet  clover  Imelilotus  alba) .  This  is  a  rank-growing  leguminous 
plant,  frequently  reaching  a  height  of  four  to  six  feet.  When  young  it 
markedly  resembles  alfalfa,  but  it  can  easily  be  distinguished  by  its  char- 
acteristic odor  when  cut  or  bruised,  as  by  rubbing  between  the  hands. 


*In  this  connection  attention  is  called  to  the  fact  that  the  so-called  "spot  dis- 
ease" of  alfalfa,  which  is  not  uncommon  in  the  western  states,  especially  duiing  wet 
seasons,  became  somewhat  prevalent  in  Illinois  in  1903.  When  the  effect  of  this 
disease  becomes  marked,  the  leaves  turn  yellow  and  growth  is  retarded.  If  this 
occurs  the  alfalfa  should  be  clipped.  This  is  the  only  effective  remedy  known  to  be 
practicable.  Seeding  alfalfa  with  a  light  nurse  crop  is  gaining  favor  in  Illinois. 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  323 

As  the  sweet  clover  approaches  maturity  it  differs  very  much  from 
alfalfa.  The  sweet  clover  grows  very  tall,  and  usually  branches  from 
a  main  stem.  It  has  white  flowers  (there  is  also  a  less  common  yellow 
variety),  and  the  seeds  are  borne  in  small  round  pods  (usually  containing 
only  one  or  two  seeds  each),  arranged  on  long  slender  spikes,  each  spike 
bearing  many  pods.  The  alfalfa  commonly  grows  about  two  and  a  half 
feet  high,  with  many  stems  growing  from  the  crown  of  the  root,  especially 
after  it  is  two  or  three  years  old.  It  bears  purple  flowers  and  peculiar 
spiral-shaped  seed  pods.  Sweet  clover  is  a  biennial  plant,  dying  after 
reaching  maturity,  which  commonly  occurs  the  second  year  of  its  growth. 
Like  many  other  biennial  plants,  it  probably  often  lives  more  than  two 
years  if  not  allowred  to  produce  seed.  Alfalfa  is  a  perennial  plant,  and 
it  is  said  that  there  are  alfalfa  fields  which  have  been  cut  annually  for 
more  than  fifty  years  without  reseeding.  The  similarity  of  alfalfa  and 
sweet  clover  when  young,  and  also  the  similarity  of  the  tubercles  formed 
on  the  roots  of  each  have  long  been  noticed,  and  the  possibility  of  the 
same  bacteria  living  upon  both  plants  has  already  been  suggested  in  the 
agricultural  press. 

During  the  season  of  1903  the  writer  spent  some  time  in  the  northern 
part  of  Illinois  in  connection  with  the  general  and  detail  surveys  of 
Illinois  soil.  Many  new  fields  of  alfalfa  were  observed,  and  they  were 
carefully  examined  for  root  tubercles.  In  Winnebago  County,  where 
sweet  clover  is  very  prevalent  along  roadsides  and  in  waste  places,  it 
was  noted  that  the  abundance  of  root  tubercles  on  the  alfalfa  plants 
seemed  to  be  closely  related  to  the  presence  of  sweet  clover  in  the  vicinity, 
strongly  indicating  that  the  bacteria  which  live  upon  sweet  clover  were 
also  at  home  upon  the  alfalfa  roots.  These  indications  were  strengthened 
by  further  investigations  in  Lake  County,  especially  upon  the  Fowler 
farm,  near  Lake  Villa,  where  a  field  of  alfalfa  seeded  last  spring  without 
artificial  inoculation  was  found  to  be  thoroughly  infected  with  the  bac- 
teria, and  growing  vigorously  with  a  good  dark  green  color.  This  field 
had  a  few  sweet  clover^ 'plants  growing  in  it,  and  the  borders  of  the  field 
were  covered  with  sweet  clover.  Other  fields  of  alfalfa  seeded  in  the 
neighborhood  at  the  same  time,  but  upon  soils  where  sweet  clover  had 
not  grown  near  by,  were  apparently  complete  failures,  many  of  the  plants 
having  died  and  most  of  those  still  living  being  only  a  few  inches  high, 
very  weak,  and  yellow  or  pale  green  in  color.* 

In  order  to  obtain  more  absolute  knowledge  regarding  this  important 
subject,  a  series  of  pot  culture  experiments  has  been  carried  on  under 
controlled  conditions  in  the  pot  culture  laboratory  at  the  university. 
Five  pots  were  filled  with  sterilized  sand  which  was  practically  devoid 

*Someof  these  observations  have  already  been  reported  in  the  agricultural  press. 
(See,  for  example,  the  Breeders'  Gazette,  September  9,  1903,  page  391,  and  Sep- 
tember 16,  1903,  page  442.) 


324  BULLETIN  No.  94.  [February, 

of  plant  food.  A  supply  of  phosphorus,  potassium,  and  all  other  mineral 
elements  necessary  for  the  growth  of  plants  was  added  to  each  of  the  five 
pots,  care  being  taken  to  keep  the  sand  practically  free  of  combined 
nitrogen.  Alfalfa  seed  were  then  planted  in  each  of  the  five  pots,  and 
at  the  same  time  four  of  the  five  pots  were  inoculated  as  follows : 

Pot  No.  1. — Not  inoculated  (check  pot). 

Pot  No.  2. — Inoculated  with  bacteria  obtained  from  infected  alfalfa 
soil. 

Pot  No.  3. — Inoculated  with  bacteria  obtained  from  alfalfa  root 
tubercles. 

Pot  No.  4. — Inoculated  with  bacteria  obtained  from  infected  sweet 
clover  soil. 

Pot  No.  5. — Inoculated  with  bacteria  obtained  from  sweet  clover  root 
tubercles. 

Plate  5  clearly  shows  the  results  obtained  and  certainly  furnishes 
conclusive  proof  that  the  same  effect  is  produced  upon  the  growth  of  the 
alfalfa  whether  the  nitrogen-gathering  bacteria  used  for  the  inoculation 
are  obtained  from  alfalfa  soil,  from  alfalfa  tubercles,  from  sweet  clover 
soil,  or  from  sweet  clover  tubercles.  It  also  illustrates  the  importance 
of  bacteria  in  growing  alfalfa  as  will  be  seen  by  comparing  the  four 
inoculated  pots  with  the  uninoculated  pot,  which  is  No.  1,  on  the  left  in 
each  series  of  views.  The  upper  view  was  taken  when  the  alfalfa  plants 
were  five  weeks  old;  the  next  series  when  they  were  six  weeks  old;  the 
next,  seven  weeks  old;  and  the  lower  series  when  they  were  eight  weeks 
old,  from  the  time  of  seeding. 

A  duplicate  series  of  pots  prepared  in  exactly  the  same  manner  gave 
similar  results. 

The  infected  alfalfa  soil  was  obtained  from  a  field  of  three-year-old 
alfalfa  which  was  inoculated  when  first  seeded,  with  infected  alfalfa  soil 
obtained  from  an  old  alfalfa  field  in  Kansas.  About  one  pound  of  this 
soil  was  shaken  in  a  quart  of  water,  the  soil  allowed  to  settle,  and  some 
of  the  nearly  clear  solution  used  for  the  inoculation  of  Pot  No.  2.  The 
alfalfa  tubercles  from  which  bacteria  were  obtained  were  carefully  washed 
in  distilled  water  to  free  them  from  adhering  soil  particles,  and  then 
rubbed  up  in  distilled  water,  a  small  amount  of  this  water  being  then 
used  for  the  inoculation  of  Pot  No.  3.  The  infected  sweet  clover  soil  was 
obtained  from  a  place  by  the  roadside  where  sweet  clover  was  growing 
luxuriantly  and  well  provided  with  root  tubercles.  This  place  was  about 
two  miles  from  the  nearest  field  ever  seeded  to  alfalfa,  so  far  as  known. 
A  water  extract  from  this  soil  was  used  to  inoculate  Pot  No.  4.  The 
bacteria  from  sweet  clover  tubercles  were  obtained  in  the  same  manner 
as  those  from  alfalfa  tubercles,  and  were  used  to  inoculate  Pot  No.  5. 

From  these  investigations  we  thus  have  conclusive  evidence  that  in- 
fected sweet  clover  soil  can  be  used  for  the  inoculation  of  alfalfa  fields. 


1904.] 


NITROGEN  BACTERIA  AND  LEGUMES. 


325 


PLATE  5.    ALFALFA  :  EFFECT  OF  BACTERIA  FROM  ALFALFA  AND  FROM  SWEET  CLOVER. 

Pot  1. — No  bacteria. 
Pots  2  and  3. — Bacteria  from  alfalfa. 
Pots  4  and  5. — Bacteria  from  sweet  clover. 

The  four  series  of   photographs  were  taken  five,  six,  seven,  and  eight  weeks  from  time  of 
planting,  respectively. 

the  bacteria  of  the  two  plants  acting  the  same.  The  infected  soil  may  be 
obtained  from  any  place  where  the  sweet  clover  is  found  growing  with 
abundance  of  tubercles  on  its  roots.  The  soil  may  be  collected  to  a 
depth  of  three  or  four  inches  and  scattered  over  the  alfalfa  field  at  the 
rate  of  100  pounds  or  more  to  the  acre.  It  is  well  to  scatter  the  infected 
soil  at  about  the  time  the  alfalfa  is  seeded,  and  harrow  it  in  with  the 
alfalfa  seed,  although  it  may  be  applied  some  days  or  even  some  weeks 


326  BULLETIN  No.  94.  [February, 

before  seeding  time,  and  probably  it  would  be  all  right  to  apply  the 
infected  soil  the  fall  before,  for  it  is  known  that  the  bacteria  will  live  in 
soil  for  several  months,  even  though  the  soil  be  placed  in  sacks  and 
allowed  to  become  quite  dry. 

Investigations  have  shown  that  100  pounds  of  thoroughly  infected 
soil  to  the  acre  is  sufficient  to  produce  a  very  satisfactory  inoculation 
within  cne  year  from  the  time  it  is  applied.  Of  course,  somewhat  heavier 
applications  may  well  be  made  if  it  can  be  done  at  small  expense.  The 
infected  soil  need  not  be  applied  with  any  high  degree  of  uniformity,  but 
special  care  should  be  taken  that  the  higher  places  and  watersheds  are 
not  missed  in  scattering  it  over  the  field.  If  a  few  square  yards,  or  even 
square  rods,  should  be  missed  on  the  slopes  or  lower  land,  it  would  make 
but  little  difference,  as  the  bacteria  will  be  washed  over  such  places  from 
the  higher  land. 

After  the  soil  becomes  somewhat  dry  it  is  easily  scattered  by  hand 
from  the  wagon  or  from  a  sack  which  one  can  carry.  Sometimes  it  is 
applied  by  means  of  an  end  gate  seeder  or  a  fertilizer  drill,  or  it  could 
be  spread  by  a  manure  spreader  with  an  application  of  manure. 

The  question  naturally  arises  whether  there  is  not  danger  of  getting 
some  sweet  clover  seed  with  the  infected  sweet  clover  soil,  and  thus  of 
getting  sweet  clover  mixed  with  the  alfalfa  in  the  field. 

In  the  writer's  opinion  there  is  little  or  nothing  to  fear  in  this  matter. 
In  the  first  place,  the  amount  of  sweet  clover  seed  thus  obtained  would 
be  very  small,  probably  none  at  all,  if  one  were  careful  to  scrape  off  the 
vegetable  matter,  and  perhaps  a  half  inch  of  earth  before  collecting  the 
infected  soil  (most  of  the  bacteria  are  probably  between  one-half  inch 
and  six  inches  in  depth,  as  most  of  the  tubercles  develop  and  decay 
between  these  depths) ;  second,-  it  is  doubtful  if  a  small  amount  of  sweet 
clover  hay  would  lessen  the  value  of  alfalfa  hay  in  the  least,  for  stock 
frequently  eat  small  amounts  of  sweet  clover  of  their  own  choice  even 
when  it  is  nearly  mature,  and  if  it  is  cut  while  still  quite  immature  and 
tender  it  makes  quite  satisfactory  hay,  so  much  so  that  in  some  sections 
of  the  United  States,  particularly  in  the  South,  sweet  clover  is  regularly 
seeded  on  fields  and  cut  for  hay,  and  it  is  found  to  be  a  valuable  and 
very  nutritious  feed,  the  live  stock  eating  it  in  large  quantities,  and  with 
apparent  relish,  after  they  have  acquired  a  taste  for  it;  third,  sweet  clover 
is  not  known  as  a  bad  weed  in  the  fields  or  meadows,  even  where  it  has 
been  a  common  roadside  plant  for  many  years,  and,  being  naturally  a 
biennial  plant,  if  it  were  cut  down  every  five  or  six  weeks,  as  we  commonly 
cut  alfalfa  during  the  season,  it  would  almost  certainly  die  out  after  a 
few  years  while  alfalfa,  a  perennial  plant,  would  continue  to  live. 

Only  one  instance  has  come  to  the  writer's  attention  where  alfalfa 
has  been  growing  for  several  years  with  sweet  clover  growing  in  the 


1904.]  NITROGEN  BACTERIA  AND  LEGUMES.  327 

field  or  fence  rows  beside  it.     This  is  on  the  farm  of  Mr.  D.  S.  Mayhew, 
of  Mercer  County,  Illinois,  who  writes  as  follows  regarding  the  matter : 

"Will  say  that  the  sweet  clover  has  made  no  headway  in  my  meadow,  as  it 
did  not  go  to  seed,  on  account  of  my  cutting  it  so  often.  The  sweet  clover  got 
into  the  alfalfa  in  the  seed  when  I  sowed  it.  I  do  not  think  it  will  do  any  harm 
in  a  meadow,  but  I  believe  it  would  do  harm  in  a  pasture  if  it  wasn't  cut  down* 
as  stock  will  not  eat  the  sweet  clover." 

Of  course  if  sweet  clover  should  get  into  the  field  and  persist  in  grow- 
ing, and  if  it  were  found  to  injure  the  alfalfa  appreciably  or  markedly, 
we  can  always  resort  to  plowing  the  ground  up  and  growing  corn  or  other 
crops,  thus  obtaining  some  benefit  from  the  leguminous  crop  for  its  fer- 
tilizing value,  and  at  the  same  time  completely  eradicating  the  sweet 
clover,  but  leaving  the  soil  well  infected  with  alfalfa  bacteria  ready  to 
serve  in  case  alfalfa  should  be  again  seeded  within  a  few  years. 

CONCLUSIONS. 

In  general  agriculture  in  Illinois,  whether  it  be  grain  farming  or 
ordinary  livestock  farming,  the  growing  of  legumes  is  absolutely  essen- 
tial as  a  part  of  any  economic  system  which  shall  maintain  the  fertility 
of  the  soil;  and  for  the  successful  growing  of  legumes  the  presence  and 
assistance  of  the  proper  species  of  nitrogen-gathering  bacteria  are  also 
absolutely  essential.  These  facts  being  granted,  it  certainly  follows  that 
when  sowing  any  legume  on  land,  where  the  same  legume  has  never  been 
grown  before,  or  perhaps  where  it  has  not  been  successfully  grown  within 
recent  years,  we  should  always  consider  the  matter  of  inoculation;  and, 
unless  there  is  good  reason  to  believe  that  the  soil  has  been  inoculated 
by  the  washing  from  other  higher  lying  land  where  these  bacteria  are 
known  to  be  present  or  by  applications  of  manure  made  from  that 
legume,  or  by  some  other  such  incidental  means;  or  unless  there  is  evi- 
dence that  the  bacteria  are  carried  with  the  seed  in  sufficient  quantity 
to  effect  a  satisfactory  inoculation  (as  appears  to  be  the  case  with  the 
cowpea),  then  we  should  inoculate  the  soil  directly  with  the, specific 
bacteria  required  by  the  legume  which  we  desire  to  grow. 

While  some  Illinois  soils  are  becoming  deficient  in  phosphorus  and 
in  lime,  especially  in  the  southern  part  of  the  state,  and  while  phos- 
phorus* and  ground  limestone  can  be  applied  to  such  soils  with  marked 
benefit  and  profit,  especially  for  the  growing  of  legumes,  there  is  abun- 
dant evidence  that  one  of  the  dominant  causes  for  the  failure  or  unsatis- 
factory growth  of  some  of  our  most  valuable  legumes,  and  on  some  soils 
the  sole  cause  of  failure,  is  the  absence  of  the  proper  nitrogen-gathering 
bacteria. 

There  is  no  reason  to  believe  that  any  of  the  different  species  of 
nitrogen-gathering  bacteria  will  live  in  the  soil  for  more  than  a  few 

*  Steamed  bone  meal  is  the  most  economical  and  satisfactory  form  of  phosphorus 
for  use  on  Illinois  soils,  unless  ground  rock  phosphate  (not  acid  phosphate)  shall 
prove  to  be  still  more  economical.  Experiments  to  determine  this  are  in  progress. 


328  BULLETIN  No.  94.  [February,  1904. 

years*  in  the  entire  absence  of  any  legume  upon  which  they  naturally 
live,  and  the  accumulating  evidence  strongly  indicates  that  the  bacteria 
which  are  present  in  places  in  our  soils,  such  as  the  red  clover  bacteria, 
now  found  abundantly  in  many  places  in  the  state,  especially  in  northern 
and  central  Illinois,  the  cowpea  bacteria  more  common  in  southern 
Illinois,  and  the  alfalfa  or  sweet  clover  bacteria,  which  are  becoming 
prevalent  in  some  sections — that  all  these  have  been,  and  are  being, 
gradually  introduced  and  extended  almost  entirely  by  mere  chance. 
Of  course  if  the  wagon-wheel,  which  carries  the  mud  along  the  road, 
carries  with  it  sweet  clover  seed  from  one  place  to  another,  it  may  also 
carry  the  sweet  clover  bacteria  which  live  on  the  sweet  clover  roots. 

It  now  seems  absurd  to  suppose  that  there  were  red  clover  bacteria 
in  Illinois  soil  before  red  clover  itself  was  grown  on  Illinois  soil,  unless 
the  same  bacteria  live  also  upon  some  other  legume  which  was  native  to 
our  soils.  There  is  some  evidence  that  the  vetch  bacteria  are  native  to 
our  soil,  possibly  living  upon  the  native  wild  vetches.  At  any  rate, 
tubercles  commonly  develop  on  vetch  roots  without  artificial  inoculation. 
Investigations  are  in  progress  to  ascertain  whether  the  notorious  failure 
of  crimson  clover  in  Illinois  may  not  be  due  in  part,  at  least,  to  the  ab- 
sence of  the  proper  bacteria.  (It  has  been  stated  by  some  writers  that 
the  bacteria  of  crimson  clover  and  those  of  red  clover  are  identical,  but 
we  already  have  some  reason  to  doubt  the  accuracy  of  this  statement.) 

*  Just  how  long  the  bacteria  will  live  in  a  soil  without  a  leguminous  crop  upon 
which  they  can  feed  is  not  definitely  known.  Certainly  they  live  for  two  or  three 
years,  but  probably  not  more  than  five  or  six  years.  Further  investigation  is  needed 
to  establish  the  length  of  time  the  different  kinds  of  bacteria  may  remain  in  the 
soil  under  different  conditions. 


UNIVERSITY  OF  ILLINOIS-URBANA 


